WO2022270872A1 - Procédé commercial de purification de vésicules extracellulaires bactériennes de grande pureté - Google Patents

Procédé commercial de purification de vésicules extracellulaires bactériennes de grande pureté Download PDF

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WO2022270872A1
WO2022270872A1 PCT/KR2022/008791 KR2022008791W WO2022270872A1 WO 2022270872 A1 WO2022270872 A1 WO 2022270872A1 KR 2022008791 W KR2022008791 W KR 2022008791W WO 2022270872 A1 WO2022270872 A1 WO 2022270872A1
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extracellular vesicles
bacterial
bacteria
precipitation
derived extracellular
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Korean (ko)
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이창진
송성현
김시원
이태룡
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(주)로제타엑소좀
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Priority to EP22828719.9A priority Critical patent/EP4361252A1/fr
Priority to CN202280056840.2A priority patent/CN117836401A/zh
Publication of WO2022270872A1 publication Critical patent/WO2022270872A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention provides an efficient method for mass-purifying bacterial-derived extracellular vesicles (EVs) with high purity. Specifically, the present invention relates to a method for rapidly and conveniently separating and purifying high-purity bacteria-derived extracellular vesicles from a large amount of bacterial cell culture using calcium cations or cobalt cations.
  • Bacterial-derived extracellular vesicles are vesicles with a size of approximately 10 nm to 1000 nm, and contain various biologically active substances of origin bacteria such as proteins, lipids, genetic materials (DNA, RNA), and virulence factors. . Bacterial-derived extracellular vesicles can be classified into gram-negative bacteria-derived extracellular vesicles and gram-positive bacteria-derived extracellular vesicles, and extracellular vesicles secreted from gram-negative bacteria are outer membrane endoplasmic reticulum ( Also known as the outer membrane vesicle (OMV).
  • OMV outer membrane endoplasmic reticulum
  • bacteria-derived extracellular vesicles function as information carriers such as the transfer of proteins or genetic materials between homologous bacteria, eliminate competing bacteria, contribute to the enhancement of bacterial survival, and deliver pathogenic toxins to the host, thereby contributing to bacterial infectious diseases. It is known.
  • bacteria-derived extracellular vesicles are as small as nanometers in size, and in bacterial cell cultures, other protein particles (e.g., flagellin, Flagellin protein particles) and nucleic acid particles, making it difficult to isolate pure bacterial-derived extracellular vesicles from large quantities of bacterial cell cultures while intactly preserving the structure and function of the extracellular vesicles.
  • protein particles e.g., flagellin, Flagellin protein particles
  • Density separation method, particle size separation method, immunoaffinity separation method, etc. are known as conventional separation techniques for bacteria-derived extracellular vesicles. For example, ultra-centrifugation (Momen-Heravi et al., Methods Mol Biol ., 1660: 25-32, 2017), size exclusion (Gamez-Valero et al., Sci. Rep . 6: 33641, 2016), immunoaffinity isolation, microfluidics Chip technology (microfluidics chip) and polymer precipitation (polymer precipitation) (Niu et al., PLoS ONE , 0186534, 2017), etc. are used.
  • the density difference separation method is a method of separation using the density of the liquid, which is about 1.0, while the density of the extracellular vesicles is about 1.13 to 1.19.
  • the ultracentrifugation method is the most widely used, and the principle is simple, so it is the most reliable separation method. to be.
  • the separation step is complicated, requiring a lot of labor and time, expensive equipment, and the maximum amount of fluid allowed at one time is only 0.6 L, and the yield and purity are extremely low. However, it is not suitable for obtaining large amounts of bacteria-derived extracellular vesicles.
  • the size-specific exclusion method is a method of filtering out extracellular vesicles using a filter having a pore size smaller or similar to that of bacterial-derived extracellular vesicles, and has the advantage of fast separation time and high purity of extracellular vesicles as it does not require reaction time.
  • the yield after separation is low because the ratio of extracellular vesicles sticking to or escaping from the filter is high.
  • the immunoaffinity separation method is a method of separating antibodies by attaching them to bacterial-derived extracellular vesicles, and has the advantage of very high specificity.
  • the polymer precipitation method is a method of eluting and precipitating by reducing the solubility of bacteria-derived extracellular vesicles by adding a hydrophilic polymer such as polyethylene glycol (PEG), and has the advantage of being able to separate with little centrifugal force.
  • a hydrophilic polymer such as polyethylene glycol (PEG)
  • PEG polyethylene glycol
  • SBI's product (ExoBacteria TM OMV Kit) is commercially available as an OMV isolation kit for Gram-negative bacteria.
  • the product using the OMV affinity resin column has a separation capacity of only 30 ml per column, and is not suitable for mass production.
  • Non-Patent Document 1 Momen-Heravi et al., Methods Mol. Biol., 1660: 25-32, 2017
  • Non-Patent Document 2 Gamez-Valero et al., Sci. Rep. 6:33641, 2016
  • Non-Patent Document 3 Niu et al., PLoS ONE, 0186534, 2017
  • An object of the present invention is to provide a method for simply and rapidly isolating and purifying high-purity bacteria-derived extracellular vesicles from a large amount of bacterial cell culture.
  • a further object of the present invention is to provide a high-purity and high-efficiency purification method applicable to bacterial-derived extracellular vesicles that solves the problem of isolating other protein particles or nucleic acid particles together in addition to bacterial-derived extracellular vesicles when using conventional separation techniques. is to provide
  • the present invention was completed based on unexpected research results that calcium cations or cobalt cations enable high-purity purification of bacterial-derived extracellular vesicles and are useful for mass processing.
  • the bacteria-derived extracellular vesicles and calcium cation or cobalt cation bind to form an insoluble complex.
  • the calcium cation or cobalt cation effectively inhibits the co-precipitation of other protein particles or nucleic acid particles derived from bacteria other than the bacteria-derived extracellular vesicles, thereby separating and purifying the bacterial-derived extracellular vesicles from the bacterial cell culture with high purity.
  • the formed bacterial-derived extracellular vesicle-calcium cation complex or the bacterial-derived extracellular vesicle-cobalt cation complex is separated by various methods such as centrifugation or precipitation by gravity, and then the calcium cation or cobalt cation is desorbed from the complex, thereby desorbing the bacterial-derived extracellular vesicle-cobalt cation complex.
  • Extracellular vesicles can be easily purified with high efficiency.
  • the present invention comprises the steps of (a) adding calcium cations or cobalt cations to a bacterial cell culture; (b) forming an insoluble complex by reacting the bacterial-derived extracellular vesicles contained in the bacterial cell culture with calcium cations or cobalt cations; (c) isolating a complex of bacterial-derived extracellular vesicles and calcium cations or a complex of bacterial-derived extracellular vesicles and cobalt cations from the bacterial cell culture; and (d) separating calcium cations or cobalt cations from the complex to purify bacteria-derived extracellular vesicles.
  • the method of the present invention is not limited to the throughput of bacterial cell cultures, so large-scale processing is possible. Therefore, the method of the present invention can simply and quickly purify a large amount of high-purity bacteria-derived extracellular vesicles by processing a large amount of bacterial cell culture, and can be implemented on a commercial scale.
  • bacteria-derived extracellular vesicles include outer membrane vesicles, shedding vesicles, exosomes, ectosomes, microvesicles, microparticles, It may be referred to as nanovesicles and the like, and it is understood that all of them are included in the scope of the bacterial-derived extracellular vesicles of the present invention.
  • Gram-negative bacteria-derived extracellular vesicles include outer membrane-derived extracellular vesicles, inner membrane-derived extracellular vesicles, and inner/outer membrane-derived extracellular vesicles (existing both outer and inner membrane components, or having an inner membrane within the outer membrane, conversely, an outer membrane within an inner membrane). forms with , etc.) are included, and these are also understood to be included in the bacterial-derived extracellular vesicles of the present invention.
  • the "bacterial cell culture” of the present invention refers to a culture medium in which bacteria (including genetically modified bacteria) cells are cultured in a culture medium.
  • the culture medium is not particularly limited as long as it is used for culturing bacterial cells, and "natural medium” using materials of unknown composition, such as serum and tissue extracts, and "chemical composition” prepared only with substances having clear composition and chemical properties. Badge” is included.
  • the bacterial cell culture may preferably be cultured in a chemical composition medium.
  • the chemical composition medium that can be used in the present invention may be selected from the group consisting of M9 medium, DMEM medium (Dulbecco's modified Eagle's medium) and RPMI 1640 medium (Roswell Park Memorial Institute medium 1640), but is not limited thereto.
  • bacteria include naturally occurring gram-negative and gram-positive bacteria and genetically engineered bacteria.
  • the genetically engineered bacteria include bacteria transformed to weaken the toxicity of the extracellular vesicles, and examples thereof include bacteria in which an endotoxin producing gene is deleted or modified.
  • it may be a gram-negative bacterium transformed to reduce the toxicity of lipopolysaccharide or a gram-positive bacterium transformed to reduce the toxicity of lipoteichoic acid.
  • the gene ( msbB ) encoding lipid A acyltransferase which is a lipid component of lipopolysaccharide, has been transformed to be deficient ( ⁇ msbB ) bacteria, most preferably ⁇ msbB transformed E. coli, but is limited thereto no.
  • the concentration of calcium cation or cobalt cation is 1 to 1000 mM, preferably 1 to 500 mM, 1 to 100 mM, 5 to 100 mM, 5 to 50 mM, 5 to 20 mM , 5 to 15 mM, or 5 to 10 mM. Particularly preferably, the concentration of calcium cation or cobalt cation is 5 to 20 mM.
  • the concentration of calcium cation or cobalt cation is less than 5 mM, the precipitation of bacterial-derived extracellular vesicles is not sufficient, and if the concentration of calcium cation or cobalt cation is 20 mM or more, even if the concentration of cation is further increased, the bacteria-derived extracellular vesicles obtained The amount does not increase proportionately.
  • the method of adding calcium cations or cobalt cations includes a method of adding a solution containing calcium cations or cobalt cations to a bacterial cell culture and a method of dissolving by adding in solid form, It is not particularly limited as long as it can react with the extracellular vesicles in the bacterial cell culture in a positive state.
  • the extracellular vesicles specifically bind with cations to form insoluble extracellular vesicle cation complexes, and since the insoluble complexes sink by gravity, they can be easily separated.
  • bacterial cell cultures contain a large amount of impurities, such as many bacterial-derived protein particles or nucleic acid particles, which have similar mass, density, size, or charge to bacterial-derived extracellular vesicles. During precipitation, the impurity particles co-precipitate together and are likely to be contaminated.
  • the impurity particles include flagellin protein particles or nucleic acid particles derived from bacterial flagellum.
  • calcium cation can effectively inhibit the co-precipitation of impurity protein particles such as flagellin or nucleic acid particles derived from bacteria when forming and precipitating insoluble complexes with bacterial extracellular vesicles.
  • the step (step (c)) of separating an insoluble complex of bacteria-derived extracellular vesicles and calcium cations or an insoluble complex of bacteria-derived extracellular vesicles and cobalt cations from a bacterial cell culture The method is not particularly limited as long as it is used to separate insoluble substances from samples containing soluble substances, and centrifugation, ultracentrifugation, filtration, ultrafiltration, gravity, dialysis, sonication, density gradient, and size exclusion. A method such as a law may be selected, but is not limited thereto.
  • filtration, centrifugation or ultrafiltration may be used in step (c) of the present invention.
  • the method used in the step (step (d)) of purifying the bacterial-derived extracellular vesicles by separating the calcium cation or cobalt cation from the complex is It is not particularly limited as long as it is a method capable of separating only bacterial-derived extracellular vesicles from the complex by removing the specific binding state of the cation, and various methods or conditions that can be understood by those skilled in the art to which the present invention pertains can be applied.
  • step (d) may use a method of adding a chelating agent to the separated bacterial-derived extracellular vesicles and calcium cation complex or the bacterial-derived extracellular vesicles and cobalt cation complex.
  • chelate agents refers to ions, molecules or atomic groups including two or more coordinating atoms that form a stable chelate complex by coordinating with metal ions, and tridentate ligands ( They are called tridentrate ligand, tetradentrate ligand, pentadentrate ligand, and hexadentrate ligand.
  • the chelating agent of the present invention is iminodiacetic acid (IDA, iminodiacetic acid), nitrilotriacetic acid (NTA, nitrilotriacetic acid), tris (carboxymethyl) ethylenediamine (TED, tris- (carboxymethyl) ethylenediamine), ethylenediamine, Ethylenediamine tetraacetate (EDTA), alkylenediamine triacetic acid, diethylenetriaminepentaacetic acid (DTPA), ethylene glycol bis(beta-aminoethyl ether)-N,N, N',N'-tetraacetic acid (EGTA, ethylene glycol-bis( ⁇ -aminoethyl ether)-N,N,N',N'-tetraacetic acid), phosphoserine and 1,4,7-triazo At least one may be selected from the group consisting of cyclononane (TACN, 1,4,7-triazocyclononane).
  • step (d) is a method of changing the pH value of a solution containing a complex of separated bacterial-derived extracellular vesicles and calcium cations or a complex of bacterial-derived extracellular vesicles and cobalt cations.
  • the step (d) is imidazole (imidazole), histidine in a solution containing a complex of separated bacterial-derived extracellular vesicles and calcium cations or a complex of bacterial-derived extracellular vesicles and cobalt cations.
  • a complex of separated bacterial-derived extracellular vesicles and calcium cations or a complex of bacterial-derived extracellular vesicles and cobalt cations histidine
  • EDTA ethylenediamine tetraacetate
  • step (d) of the present invention may apply a buffer of pH 10 or less, 0 to 5 M NaCl, 0 to 2 M imidazole, 0 to 2 M metal chelating agent, or a combination of the above conditions, Not limited to this.
  • the method of the present invention further comprises a pretreatment step of the bacterial cell culture prior to adding calcium cations or cobalt cations to the bacterial cell culture (i.e., prior to step (a)).
  • the pretreatment step of the present invention concentrates the bacterial cell culture to reduce the amount of sample to be input to the subsequent purification step, and at the same time removes bacterial cells or other protein particles or nucleic acid particles derived from bacteria from the non-purified bacterial cell culture.
  • a partial purification step for the purification centrifugation, filtration, ultrafiltration, size exclusion, desalting column chromatography, size exclusion chromatography, ion exchange chromatography, affinity chromatography, polymer precipitation, salt precipitation, organic solvent precipitation
  • At least one method may be selected from the method, the aqueous two-phase method, and the enzyme treatment method, but is not limited thereto.
  • centrifugation, filtration, polymer precipitation or salt precipitation may be used in the pretreatment step, and tangential flow filtration (TFF) is most preferred.
  • the method of the present invention may further include post-processing the purified bacterial-derived extracellular vesicles after step (d).
  • the post-processing step of the present invention is an additional purification step of purified bacterial-derived extracellular vesicles, which include centrifugation, filtration, ultrafiltration, dialysis, sonication, density gradient method, size exclusion method, desalting column chromatography, and size exclusion.
  • At least one method may be selected from among chromatography, ion exchange chromatography, affinity chromatography, polymer precipitation method, salt precipitation method, organic solvent precipitation method, aqueous two-phase method, and enzyme treatment method, but is not limited thereto.
  • ultrafiltration, dialysis, size exclusion, size exclusion chromatography, ion exchange chromatography, polymer precipitation or salt precipitation may be used, and ultrafiltration or size exclusion is particularly preferred. .
  • the method of the present invention further comprises a filtration step for removing protein particles or nucleic acid particles other than bacteria-derived extracellular vesicles before or after the precipitation step by calcium cation or cobalt cation. can do.
  • the method of the present invention may further include an enzyme treatment step for removing bacterial-derived nucleic acid particles before or after the precipitation step by the calcium cation or cobalt cation.
  • the enzyme is not particularly limited as long as it can be used to degrade nucleic acid particles from bacterial cell culture, and for example, benzonase and the like can be used, but is not limited thereto.
  • ion exchange chromatography may be preferably performed in combination with Benzonase or after Benzonase treatment.
  • the mass purification method of bacterial-derived extracellular vesicles of the present invention can be used in combination with conventional separation techniques to maximize separation efficiency, if necessary.
  • the method for mass purification of bacterial-derived extracellular vesicles can efficiently remove bacterial-derived protein particles or nucleic acid particles such as flagellin, and is not exposed to an extreme environment during the separation process, so that the bacterial-derived extracellular vesicles It can be effectively purified while preserving its structure and function.
  • the method of the present invention is not limited in the throughput of bacterial cell cultures, so it is possible to obtain a large amount of bacterial-derived extracellular vesicles by processing a large amount of bacterial cell cultures, and does not require expensive equipment or materials such as antibodies, Since high yield and purity can be achieved, high-purity bacterial-derived extracellular vesicles can be easily obtained on a commercial scale.
  • bacteria-derived extracellular vesicles obtained by the method of the present invention using calcium cations are more advantageous for development as pharmaceuticals for humans.
  • Figure 1a is a photograph of total protein staining and OmpA and FliC western blot showing impurities contained in a cell-free bacterial cell culture.
  • Figure 1b is a graph showing the results of HPLC analysis showing impurities contained in cell-free bacterial cell cultures.
  • Figure 2a is a picture of total protein staining and OmpA and FliC western blots showing the purification ability of bacterial EVs by precipitation using 6 types of metal cations and polymers (PEG).
  • Figure 2b is a graph showing the results of HPLC analysis of bacterial EV precipitated isolates using 6 types of metal cations.
  • Figure 2c is a graph showing the results of HPLC analysis after removing nucleic acids by treating bacterial EV precipitates using 6 types of metal cations with benzonase.
  • 3a and 3b are graphs showing HPLC analysis results of EV precipitates obtained by treatment with various concentrations of calcium.
  • Figure 3c is a photograph of total protein staining of EV precipitates obtained by treatment with various concentrations of calcium or PEG.
  • Figure 4 shows the results of SEC-HPLC (a), DLS (b) and NTA (c) analysis of bacterial EV obtained by carrying out the purification method of the present invention on a pilot scale 50 liter culture.
  • FIG. 5 is an investment electron micrograph (a), SDS-PAGE analysis (b), OmpA western blot (c), and a graph (d) showing the results of nano flow cytometry of the bacterial EVs obtained in FIG. 4 .
  • the E. coli w3110 (msbB mutant) culture was centrifuged at 6000 xg for 20 minutes to remove precipitated cells, and the supernatant was concentrated 10-fold by passing through a tangential flow filter (TFF).
  • 1 mL of the cell-free cell culture enriched with 10X was incubated on ice and at 37° C. for 30 minutes, and then centrifuged (12000 xg , 10 minutes).
  • 1 mL of cell-free cell culture concentrated at 10X was filtered through a 0.2 ⁇ m filter, and the obtained filtrate was centrifuged (12000 xg , 10 minutes).
  • the pellet obtained after centrifugation was suspended in HBS buffer solution and then subjected to protein analysis and HPLC analysis, and the results are shown in FIGS. 1a and 1b.
  • each sample was subjected to SDS-PAGE, and then a blot was prepared through electrotransfer on a PVDF membrane. After reacting the prepared blot with an in-house prepared Rabbit polyclonal anti-FliC or Rabbit polyclonal anti-OmpA antibody solution for more than 4 hours, the blot was washed, and the secondary antibody, HRP-conjugated anti-rabbit IgG antibody (Santa Cruz) solution and After reacting for 1 hour, it was washed. Thereafter, a luminescent reaction was induced with an ECL (Enhanced chemiluminescence, Thermo Scientific) solution to detect a luminescent signal of FliC or OmpA protein.
  • ECL Enhanced chemiluminescence, Thermo Scientific
  • the absorbance of the 260 nm, 280 nm, and 450 nm wavelengths of the solution eluted after sample injection was simultaneously recorded in real time to obtain an absorbance chromatogram and analyzed it.
  • Various particles, including bacteria-derived extracellular vesicles, peaks are detected at about 4.9 - 5.2 minutes under the corresponding column and elution conditions, and the peaks eluted after that are small non-particulate materials composed of various substances (these non-particulate matter The substance can be easily removed by dialysis, etc.).
  • the particulate impurities may be products of cell death as very large particles, may be modified inactivated extracellular vesicles, may be precipitated together with EVs during EV precipitation separation, and OmpA may be obtained after final purification of EVs. Since it may be difficult to distinguish from EV particles having EVs, it is preferable to remove them through filtration or the like before or after the EV precipitation step. In subsequent examples of the present invention, EVs were removed prior to the precipitation step.
  • Example 2 Bacterial EV isolation and purification efficacy test using various precipitation methods
  • Example 1 After passing the 10X concentrated cell-free cell culture of Example 1 through a 0.2 ⁇ m filter, 25 mM or 12% PEG6000 of 6 metal cations (CA, CO, CU, MN, NI, ZN) was added, respectively. After standing for 10 minutes, centrifuging at 12000 xg for 10 minutes to obtain a pellet, and protein analysis and HPLC analysis were performed on the pellet as in Example 1, and the results are shown in FIGS. 2A to 2C.
  • 6 metal cations CA, CO, CU, MN, NI, ZN
  • NI has a relatively low OmpA signal, resulting in low EV yield efficiency.
  • CA, CO, and MN were excellent in terms of EV yield excluding other impurities.
  • HPLC results also show similar results. That is, in the case of CA, CO, and MN, the concentration of impurities is relatively low, and in the case of NI, it can be seen that the peak signal corresponding to EV around 5 minutes is weak.
  • Example 3 EV purification efficiency test according to calcium concentration
  • the isolation and purification efficiencies of bacterial EVs were compared using various concentrations of calcium (CA).
  • Example 2 After passing the 10X concentrated cell-free cell culture of Example 1 through a 0.2 ⁇ m filter, calcium cations of various concentrations were added, left for 10 minutes, and centrifuged at 12000 xg for 10 minutes to obtain a pellet. HPLC analysis and protein analysis were performed in the same manner as in Example 1, and the results are shown in FIGS. 3A to 3C.
  • Example 4 Mass production and purification of bacterial extracellular vesicles (EV)
  • E. coli BL21(DE3) ⁇ msbB strain was cultured for 16 hours under aerobic conditions using a 75 liter fermentor system, and then cells were removed by centrifugation at 6,000 xg for 20 minutes. A culture medium was prepared. After filtration of the culture solution with a 0.2 ⁇ m filter, a 10-fold concentrated culture solution was produced through a membrane filter of 100 kDa MWCO (Molecular weight cut-off) and tangential induction ultrafiltration. The concentrated culture solution was inactivated by adding 50 ⁇ M of phenylmethylsulfonyl fluoride (PMSF) and shaking for 30 minutes to inactivate proteolytic enzymes.
  • PMSF phenylmethylsulfonyl fluoride
  • Bacterial EVs were selectively precipitated by adding a buffer solution (pH 7.2) containing 25 mM CaCl 2 to the culture medium and reacting with cold shaking for 1 hour. After harvesting the precipitate by centrifuging the reactant at 13,000 xg for 40 minutes, it was dissolved by shaking in a buffer solution (pH 7.2) containing a chelating agent, and filtered through a 0.2 ⁇ m filter.
  • the solution was placed in a dialysis membrane of 100 kDa MWCO for the exchange of non-particulate contaminants and buffers having a molecular weight smaller than bacterial EV, and the dialysis solution was replaced 5 times for a total of 18 hours at 4 ° C. to further purify the bacterial extracellular vesicles.
  • a large amount of bacterial EVs with high yield and purity can be purified from a large amount of bacterial culture without expensive equipment or materials such as antibodies.
  • purified EVs are not exposed to extreme environments, they can be effectively purified while well preserving the structure and function of bacterial-derived extracellular vesicles.

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Abstract

La présente invention concerne un procédé de purification en masse de vésicules extracellulaires bactériennes de grande pureté et, plus particulièrement, la présente invention concerne un procédé permettant d'isoler et de purifier rapidement et commodément des vésicules extracellulaires bactériennes de grande pureté à partir d'une grande quantité de produit de culture cellulaire bactérienne au moyen de calcium ou de cobalt. Le procédé de purification de la présente invention est approprié pour obtenir des vésicules extracellulaires bactériennes de grande pureté à l'échelle commerciale en traitant une grande quantité de produit de culture cellulaire bactérienne et, en particulier, en utilisant du calcium inoffensif pour le corps humain, il est plus avantageux pour purifier les vésicules extracellulaires bactériennes à utiliser dans un médicament destiné au corps humain.
PCT/KR2022/008791 2021-06-21 2022-06-21 Procédé commercial de purification de vésicules extracellulaires bactériennes de grande pureté WO2022270872A1 (fr)

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EP22828719.9A EP4361252A1 (fr) 2021-06-21 2022-06-21 Procédé commercial de purification de vésicules extracellulaires bactériennes de grande pureté
CN202280056840.2A CN117836401A (zh) 2021-06-21 2022-06-21 高纯度细菌细胞外囊泡的商业纯化方法

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KR1020210080108A KR102413278B1 (ko) 2021-06-21 2021-06-21 박테리아 유래 고순도 세포밖 소포체의 상업적 정제 방법

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